1. Field of the Invention
The present application relates generally to the detection of molecules, and more particularly, to electrochemiluminescent molecules.
2. Description of the Related Art
Electrochemiluminescence (ECL) is a phenomenon in which a species subjected to a potential at an electrode emits electromagnetic radiation, typically, visible light. A number of ECL-based assays use a bipolar electrode, that is, an electrode not electrically connected to an external circuit. This type of electrode is also known as a floating electrode. In this type of device, an external electric field is applied to an electrolyte containing the bipolar electrode. The external electric field generates anodic and cathodic regions on the floating electrode with respect to the surrounding electrolyte, hence the term bipolar electrode. Published patent applications WO 99/63347 and WO 00/03233, the disclosures of which are incorporated by reference, both disclose ECL-assays using bipolar electrodes.
An ECL detection scheme for capillary electrophoresis (CE) using a bipolar electrode is reported in Arora et al. “A Wireless Electrochemiluminescence Detector Applied to Direct and Indirect Detection for Electrophoresis on a Microfabricated Glass Device” Anal. Chem. 2001, 73, 3282-3288, the disclosure of which is incorporated by reference. In one example, the ECL reaction involves an electron transfer from electrochemically generated tripropylamine (TPA) radicals to tris-(2,2′-bipyridyl)ruthenium+3  (TBR), which radiates at λmax=610 nm. A detection limit of 5×10−13 M (S/N=3) in an effective volume of 100 nL in a small volume electrochemical cell corresponds to the detection of 30,000 TBR molecules as reported in Arora et al. “Sub-microliter Electrochemiluminescence Detector—A Model for Small Volume Analysis Systems” Anal. Commun. 1997, 34, 393-395, the disclosure of which is incorporated by reference.
ECL-based detection in CE has a number of advantages over other detection methods, for example, fluorescence detection. First, no laser excitation source is required because the method is not fluorescence-based. Second, the optical system is simpler and cheaper because the electrode provides a built-in optical alignment. The photodetector is simply aligned with the bipolar electrode. Third, the ECL detection is more sensitive, with no substrate fluorescence or excitation source background. Finally, ECL is automatically initiated by the electric field used to perform CE because the electric field generates the local potential difference at the bipolar electrode.
One problem with an ECL detection system is that electrolysis, for example of water, forms bubbles on and around the bipolar electrode, which distort the bands of the analyte or even occlude the CE channel completely. Where the medium is water, the bubbles are hydrogen and/or oxygen. In other media, the bubbles have other compositions, as is known in the art. Although bubble formation at the electrode is particularly problematic for ECL assays in confined volumes such as CE detectors, the problem is present in all ECL assays using bipolar electrodes. A second problem is that charged species, for example, ECL-active species, accumulating on or around the bipolar electrode increase the background light signal, thereby reducing the sensitivity of the system. The bipolar electrode locally “shorts” the electric field, which slows the migration of charged species in the vicinity of the bipolar electrode, thereby allowing charged species to accumulate on or around the bipolar electrode.